RESUMO
The long-term goal of this study is a training system that can simulate medical cases and advise physicians based on quantitative evaluation of neonatal resuscitation. In this paper, we designed and manufactured a neonatal airway management simulator for quantitative evaluation of tracheal intubation. This robotic simulator is equipped with 25 sensors of 6 types, which detect motions that lead to complications, inside the manikin replicated a neonate. A performance experiment of the developed sensor and an evaluation experiment with physicians were conducted. We observed that an erroneous operation in the laryngoscopy can be detected by the sensors in our simulator.
Assuntos
Intubação Intratraqueal , Ressuscitação , Manuseio das Vias Aéreas , Humanos , Recém-Nascido , Laringoscopia , ManequinsRESUMO
Human rIL-4 is able to induce the expression of low-affinity receptors for IgE (Fc epsilon RL/CD23) on resting B lymphocytes, as determined by the binding of either the anti Fc epsilon RL/CD23-specific mAb 25 or IgE. Stimulation of B cells with insolubilized anti-IgM antibody increases the number of cells expressing Fc epsilon RL/CD23 upon culturing with IL-4 and enhances the level of Fc epsilon RL/CD23 expression on these cells. Fc epsilon RL/CD23 induction is specific for IL-4 since IL-1 alpha, IL-2, IFN-gamma, B cell-derived B cell growth factor (BCGF), and a low-molecular-weight BCGF were ineffective. IFN-gamma strongly inhibited the induction of Fc epsilon RL/CD23 by IL-4.
Assuntos
Linfócitos B/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Linfocinas/farmacologia , Receptores Fc/biossíntese , Linfócitos B/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interferon gama/farmacologia , Interleucina-4 , Interfase , Receptores de IgE , Proteínas Recombinantes/farmacologiaRESUMO
We developed a novel promoter system, designated SR alpha, which is composed of the simian virus 40 (SV40) early promoter and the R segment and part of the U5 sequence (R-U5') of the long terminal repeat of human T-cell leukemia virus type 1. The R-U5' sequence stimulated chloramphenicol acetyltransferase (CAT) gene expression only when placed immediately downstream of the SV40 early promoter in the sense orientation. The SR alpha expression system was 1 or 2 orders of magnitude more active than the SV40 early promoter in a wide variety of cell types, including fibroblasts and lymphoid cells, and was capable of promoting a high level of expression of various lymphokine cDNAs. These features of the SR alpha promoter were incorporated into the pcD-cDNA expression cloning vector originally developed by Okayama and Berg.
Assuntos
DNA Recombinante , Deltaretrovirus/genética , Regulação da Expressão Gênica , Vetores Genéticos , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Vírus 40 dos Símios/genética , Animais , Linhagem Celular , Ligação de Hidrogênio , Interleucinas/genética , RNA Mensageiro/genéticaRESUMO
We have examined a prediction made from the ribosome feedback regulation model of ribosomal RNA and transfer RNA synthesis in Escherichia coli. This model proposes that non-translating "free" ribosomes act directly or indirectly as negative feedback inhibitors to regulate the transcription of rRNA and tRNA operons. One prediction of this model is that preferential inhibition of the assembly of ribosomes (without inhibiting macromolecular synthesis) should lead to a deficiency of free ribosomes which should, in turn, cause a stimulation of rRNA (and tRNA) synthesis. We have examined this prediction in vivo by causing the preferential inhibition of synthesis of certain ribosomal proteins by an overproduction of the translational repressor ribosomal protein S4. In agreement with our model, we have observed a preferential stimulation of ribosomal protein messenger RNA and rRNA synthesis under these conditions. These results suggest that ribosomes in the cellular pool, rather than incomplete ribosomal particles or free rRNA, are monitored by cells to regulate the rate of rRNA synthesis, and give further support to this proposed model.
Assuntos
Genes Bacterianos , RNA Ribossômico/biossíntese , Ribossomos/metabolismo , Proteínas de Bactérias/biossíntese , Centrifugação com Gradiente de Concentração , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , RNA de Transferência/biossíntese , Proteínas Repressoras/biossíntese , Proteínas Ribossômicas/antagonistas & inibidoresRESUMO
PIP: The human immunodeficiency virus (HIV) was introduced readily into Asia and has quickly spread between Asian states through both parenteral and sexual modes of transmission. Only 1 year after Thailand's epidemic wave among intravenous drug users (IDUs) in 1988, the virus spread to the adjacent Myanmar and Malaysia, and another year later IDUs were infected in parts of India and China bordering Myanmar. Several methods can be used to quantify the genetic diversity, divergence, or variation within or between subtypes, genotypes, or isolates. Consensus sequences, representing the most common nucleotide in the genome, are often generated for comparison. 8 subtypes A through F, H, and O have been described for HIV-1 based on the genetic similarities and differences in the env gene or viral envelope. Subtype A and D have been found primarily in central and western Africa. Subtype B is predominant in Europe, the Western hemisphere, Japan, and Australia. Subtype C has been found mostly in southern Africa, the Central African Republic, and India. Subtype E was first identified in Thailand and recently in the Central African Republic. Subtype F has been found in Romania and is a rare variant in Brazil. Isolates from Gabon and the Russian Federation were designated subtype H. An "outlier" subtype O containing 2 human and 2 chimpanzee isolates has been identified in Cameroon and Gabon. Sequencing of the relatively conserved gag gene of geographically diverse HIV-1 isolates yielded a classification with 7 subtypes A-D and F-H. Other topics discussed include genome characterization, comparison with foreign isolates, segregation by mode of transmission, and biologic properties of HIV-1 variants in Thailand; regional diversity of HIV-1 subtypes and substantial spread of HIV-2 in India; as well as HIV transmission and infections in Japan, Australia, Cambodia, China, Taiwan, Philippines, Malaysia, Myanmar, and in states created out of the former Soviet Union.^ieng
Assuntos
Infecções por HIV/epidemiologia , HIV/genética , Sequência de Aminoácidos , Ásia/epidemiologia , Genoma Viral , HIV/isolamento & purificação , Humanos , Índia/epidemiologia , Japão/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Tailândia/epidemiologiaRESUMO
We have constructed recombinant plasmids that direct the synthesis of the Mr 19 000 protein encoded by the adenovirus type 12 (Ad12) E1b region as either a native protein or a protein fused to the amino-terminal portion of the elongation factor EF-TuB in Escherichia coli cells. Using these recombinants, we could synthesize a large amount of the fused protein, while only a small amount of the native Mr 19 000 protein was produced. The failure to synthesize the native Mr 19 000 protein in E. coli cells was ascribed to inefficient translation.
Assuntos
Adenoviridae/genética , Plasmídeos , Biossíntese de Proteínas , Proteínas Virais/genética , Escherichia coli/genética , Lisogenia , Recombinação GenéticaRESUMO
We describe a robust expression of human stromal cell-derived factor-1alpha (SDF-1alpha) and SDF-1beta, the members of CXC-chemokine family, with a novel vector system based upon Sendai virus, a non-segmented negative strand RNA virus. Recombinant SDF-1alpha and SDF-1beta were detected as a major protein species in culture supernatants, reached as high as 10 microg/ ml. This remarkable enrichment of the products allowed us to use even the crude supernatants as the source for biological and antiviral assays without further concentration nor purification and will thus greatly facilitate to screen their genetically engineered derivatives.
Assuntos
Quimiocinas CXC/genética , Clonagem Molecular/métodos , Citocinas/genética , Vetores Genéticos , Respirovirus/genética , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/isolamento & purificação , Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Citocinas/isolamento & purificação , Citocinas/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Humanos , Proteínas Recombinantes/genéticaRESUMO
One of the main objectives of cancer immunotherapy is the activation and increase in number of antitumor effector cells. Recently, genetically modified tumor cell vaccines have been proposed for elicitation of antitumor effector cells. Native alpha antigen (alpha Ag) (also known as MPT59 and antigen 85B) of mycobacteria, which cross-reacts among mycobacteria species, may play an important biological role in host-pathogen interaction because it elicits various helper T-cell type 1 immune responses. To assess the induction of antitumor immune responses by alpha Ag, mouse tumor cell lines transfected with cDNA of alpha Ag from Mycobacterium kansasii were established, and the possibility of producing a tumor cell vaccine for induction of antitumor effects was explored. Transfection of tumor cell lines with an alpha Ag gene lead to primary tumor rejection and the establishment of protective immunity to nontransfected original tumor cell lines in Mycobacterium bovis bacillus Calmette-Gurin (BCG)-primed and unprimed mice. Mice immunized with tumor cell lines transfected with the alpha Ag gene showed delayed-type hypersensitivity responses in vivo and proliferative responses together with induction of interferon-gamma of spleen cells against nontransfected wild-type tumor cell lines in in vitro experiments. Moreover, immunization of mice with alpha Ag-expressing tumor cells elicited tumor-specific and cytotoxic T lymphocyte (CTL) epitope peptide-specific CD8+ CTLs. The results of this study provided evidence of the potential usefulness of alpha Ag in tumor cell vaccines.
Assuntos
Antígenos de Bactérias/genética , Vacinas Anticâncer , DNA/genética , Terapia Genética/métodos , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Animais , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mycobacterium bovis/imunologia , Mycobacterium bovis/metabolismo , Biossíntese Peptídica , Baço/citologia , Linfócitos T Citotóxicos/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
The murine AIDS (MAIDS) is a retrovirus-induced disease that shows severe immunodeficiency with abnormal lymphoproliferation in susceptible strains of mice. To clarify the antigenicity of gag gene products of the LP-BM5 defective virus, which is known as the causative virus of MAIDS, we expressed and purified the gag p12 gene product (P12) by using a baculovirus expression vector system. The P12 protein strongly stimulated the proliferation of normal C57BL/6 (B6) lymph node T-cells in vitro. Furthermore, a 25-mer synthetic polypeptide within the P12 sequence gave rise to the similar or even higher activation of T-cells. The phenotype of responding T-cells was found to be CD8+ CD44low, indicating that naive CD8+ T-cells respond against a peptide encoded within a MAIDS defective virus gag p12 gene. Finally, the expression of T-cell receptor (TcR) V beta on the responding CD8+ T-cells was analyzed. Although CD8+ T-cells with the particular V beta chains were expanded in response to the 25-mer peptide, this polypeptide does not seem to be a superantigen, since this response is MHC class I-restricted and the V beta preference is not striking. The presentation pathway of this highly antigenic polypeptide will be discussed.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Produtos do Gene gag/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Células Cultivadas , Vírus Defeituosos/química , Produtos do Gene gag/genética , Produtos do Gene gag/farmacologia , Receptores de Hialuronatos/imunologia , Vírus da Leucemia Murina/química , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Spodoptera/citologia , Células Tumorais CultivadasRESUMO
Susceptibility to HIV infection was examined in macrophages differentiated from human monocytes by macrophage colony-stimulating factor (M-CSF) or granulocyte/macrophage colony-stimulating factor (GM-CSF). The replication of macrophage-tropic human immunodeficiency virus type-1 (HIV-1), which was determined by reverse transcriptase (RT) activity, was significantly suppressed in macrophages induced by GM-CSF (GM-type macrophages) but not in those induced by M-CSF (M-type macrophages). Multinucleated giant cells were formed only in M-type macrophages after HIV infection. However, the expression of CD4 molecules on the surface of both types of macrophages was similar and the proviral DNA was detectable in cell lysates of both macrophages, although the amount of proviral DNA in M-type macrophages was higher than that in GM-type macrophages. Many steps have been defined in HIV infection and replication, such as adsorption of HIV to the cell surface, internalization of the viral core into the cytoplasm, uncoating of viral RNA, reverse transcription and integration of proviral DNA into cellular DNA, transcription and translation of proviral DNA, assembly of viral components, and budding of virus particles. Our findings suggested that the suppression of HIV-1 replication in macrophages induced by GM-CSF is mainly due to a disturbance at certain steps of replication after synthesis of the proviral DNA. Thus, the suppression of HIV replication in GM-type macrophages may provide a model of the latency of HIV infection in vivo.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Replicação Viral/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Sequência de Bases , Antígenos CD4/metabolismo , Diferenciação Celular , Células Cultivadas , Primers do DNA/genética , DNA Viral/genética , DNA Viral/metabolismo , Genes gag , Infecções por HIV/patologia , HIV-1/genética , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Microscopia Eletrônica , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/efeitos dos fármacos , Provírus/efeitos dos fármacos , Provírus/genética , Provírus/fisiologiaRESUMO
We encountered a case of HIV-1 infection in a previously healthy man, which was characterized by rapid progression to AIDS and death within 7 months in association with high levels of antigenemia throughout the clinical course and no humoral immune response for at least 6 months. Genetic changes of the third variable domain (V3) of the envelope gene of HIV-1 in serum samples were analyzed at four time points during his rapid clinical course. The nucleotide changes were confined to a maximum of three substitutions among 105 nucleotides of the V3 region. A major population of the viral clones in this patient showed one amino acid substitution from aspartic acid (a negatively charged amino acid) to lysine (a positively charged amino acid) at position 30 from the first cysteine of the V3 loop. This substitution was thought to be associated with phenotypic changes, and viruses with this sequence in the V3 region had a strong syncytium-inducing ability in MT-4 cells. It appears that the lack of a humoral immune response accelerated disease progression in our patient and a genetic change that appeared to produce a phenotypic change occurred at an early stage of the disease.
Assuntos
Síndrome da Imunodeficiência Adquirida/microbiologia , Síndrome da Imunodeficiência Adquirida/fisiopatologia , HIV-1/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Adulto , Sequência de Bases , Linhagem Celular , DNA Viral , Variação Genética , Soropositividade para HIV/microbiologia , HIV-1/imunologia , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Fatores de TempoRESUMO
PIP: Scientists wanted to identify the genetic characteristics of 2 HIV-1 subtypes in Thailand. Staff from regional laboratories of the Ministry of Public Health took blood samples from people in various high risk groups and from all regions of the country. Staff at the National Institutes of Health in Bangkok then did lymphocyte separation, DNA extraction, and virus culture. They took the extracted DNA specimens and sent them to the US Centers for Disease Control where scientists did serologic testing, polymerase chain reaction, and sequence determination. They used Kimura's method to study sequence variations. They sequenced 300 nucleotides, including the C2-V3 domains of HIV-1 envelope gene and/or hybridization. Every risk group had HIV-1 subtype A, but subtype B was mostly found in drug users. Subtype A had spread mainly among heterosexuals. The mean intraperson variation for subtypes A and B stood at 2% and 2.7%, respectively, while the interperson variation within subtype A and B stood at 3.8% and 3.7%, respectively. The mean interperson variation between subtypes A and B from different persons was 18.1%. Phylogenetic tree analysis showed that subtype B identified with about 85% of the sequence as that of the North American isolates, making it more closely related to them than to African isolates (about 75% sequence identity). On the other hand, subtype A had a GPGQ motif at the V3 crown which was common among African HIV-1 isolates. Antibodies which usually recognize HIV-1 MN strains (which have the GPGR motif) may not react wholly with the V3 loop from the Thailand subtype A viruses, thus the GPGQ motif at the V3 crown may pose a problem. Now for the first time, scientists can follow the natural history of 2 HIV-1 subtypes and determine their relative pathogenicity and transmission efficiency between adults or from mother to infant. The relative homogeneity of the HIV-1 strains in Thailand presents a theoretical advantage in designing vaccines for potential large-scale clinical trials.^ieng
Assuntos
Infecções por HIV/epidemiologia , HIV-1/classificação , Sequência de Aminoácidos , Surtos de Doenças , Feminino , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/complicações , Infecções por HIV/microbiologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Homologia de Sequência de Aminoácidos , Abuso de Substâncias por Via Intravenosa/complicações , Abuso de Substâncias por Via Intravenosa/microbiologia , TailândiaRESUMO
The envelope of human T-cell leukemia virus type I (HTLV-I) consists of two glycoproteins gp46 and p20E. Recombinant envelope proteins were produced by using an expression vector derived from insect baculovirus, Bombyx mori nuclear polyhedrosis virus. Polyhedrin fusion proteins C182, N147, and N287 contained whole region p20E, C-terminal half of gp46, and almost whole region gp46, respectively. N147 and N287 were suggested to be processed forms resulting from internal cleavage by cellular enzymes. In cultured cells and the insect larvae, C182 and N147 were produced abundantly enough to be purified to homogeneity; however, N287 was produced poorly and not purified. The purified proteins were recognized by HTLV-I-infected human sera and shown to be highly specific antigens for blood screening systems.
Assuntos
Produtos do Gene env , Vírus Linfotrópico T Tipo 1 Humano/genética , Proteínas Oncogênicas de Retroviridae/genética , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Bombyx , Linhagem Celular , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos , Cobaias , Anticorpos Anti-HTLV-I/sangue , Anticorpos Anti-HTLV-I/imunologia , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Immunoblotting , Dados de Sequência Molecular , Proteínas de Matriz de Corpos de Inclusão , Proteínas Recombinantes de Fusão/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais/imunologia , Proteínas Estruturais ViraisRESUMO
We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.
Assuntos
Soropositividade para HIV/virologia , HIV-1/classificação , HIV-1/genética , Filogenia , Recombinação Genética , Replicação Viral , Sequência de Aminoácidos , Sequência de Bases , DNA Viral , Feminino , Gana , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Reação em Cadeia da PolimeraseRESUMO
We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.
Assuntos
Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Recombinação Genética , Proteínas Virais , Adolescente , Adulto , Sequência de Aminoácidos , Feminino , Produtos do Gene gag/química , Produtos do Gene gag/genética , Antígenos HIV/química , Antígenos HIV/genética , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Humanos , Masculino , Dados de Sequência Molecular , Mianmar/epidemiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Filogenia , Análise de Sequência de DNA , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
PIP: The authors examined HIV-1 genetic variation among 19 HIV-1-infected people of mean age 34.5 years living in Accra, Akwatia, Kumasi, and Ho, in Ghana. One person was of unknown origin. Blood samples were collected between December 1993 and January 1996. 16 of the HIV-1 specimens clustered with members of subtype A, but the clustering was not supported by 70% or more of the bootstrap tests. Two samples clustered with subtype D strains, supported by 92.5% of the bootstrap trees, and one sample clustered with subtype G strains, supported by 96.2% of the bootstrap trees. For the Ghanaian specimens belonging to subtype A, interhost distances at the nucleotide level averaged 14.9%, of range 7.83-20.9%. The interhost distance between the two subtype D samples was 8.2%. A cocirculation of subtypes A, D, and G was identified in Akwatia.^ieng
Assuntos
Genes env/genética , Infecções por HIV/genética , HIV-1/genética , Adolescente , Adulto , Sequência de Aminoácidos , Feminino , Heterogeneidade Genética , Gana/epidemiologia , Proteína gp120 do Envelope de HIV/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Produtos do Gene env/metabolismo , Produtos do Gene nef/metabolismo , Produtos do Gene vpr/metabolismo , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Células HeLa , Humanos , Immunoblotting , Índia , Dados de Sequência Molecular , Fenótipo , Filogenia , Receptores CCR5/metabolismo , Análise de Sequência de DNA , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Previous serological studies have demonstrated that some 60% of intravenous drug abusers (IVDAs) in urban areas of the former South Vietnam are infected with HTLV-II. In the present report we have attempted to characterize the viruses using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of the provirus long terminal repeat (LTR) region. RFLP analysis of nine samples demonstrated that all were infected with the HTLV-IIb subtype. The HTLV-IIa subtype was not detected. Phylogenetic analysis of the nucleotide sequences demonstrated that the viruses clustered closely with HTLV-IIb isolates present in IVDAs from the New York City area. The present molecular analysis together with the previously reported absence of HTLV-II infection in North Vietnam supports the view that HTLV-II may have been introduced from the United States to this part of Asia by military personnel during the Vietnam conflict.
Assuntos
Infecções por HTLV-II/virologia , Vírus Linfotrópico T Tipo 2 Humano/genética , Filogenia , Abuso de Substâncias por Via Intravenosa/virologia , Humanos , Reação em Cadeia da Polimerase , VietnãRESUMO
To study the molecular epidemiology of HIV-1 spread in Myanmar and the interplay with the epidemic in surrounding Southeast Asian countries, we determined the HIV-1 subtypes prevailing in Myanmar. Thirty HIV-positive blood specimens were sampled in the capital city, Yangon, and an additional 459 sera were collected nationwide in 1995. Genetic subtyping based on the env C2/V3 sequence and serologic data, using a V3 peptide enzyme immunoassay (PEIA), revealed three patterns of HIV spread in different geographic regions in Myanmar: (1) in the capital city, Yangon, HIV-1 subtype B' ("Thai-B" cluster within subtype B) predominated both in IDUs and heterosexuals; (2) in the cities near the border with Thailand, including Tachelaik and Kawthaung, where heterosexual transmission is a major pathway of HIV-1 spread, HIV-1 subtype E was predominantly distributed among the commercial sex workers and heterosexuals; (3) in central and northeast Myanmar, both HIV-1 subtypes B' and E occurred in a mixed distribution, without showing any significant segregation by risk group. In addition, the PEIA data implied the occurrence of other subtype(s) in these areas. The interperson nucleotide sequence variations in env C2/V3 regions of B' and E, prevailing in Yangon, were 6.7 +/- 2.1 and 7.1 +/- 0.7%, respectively. They were similar to those levels observed in Thailand. These findings are consistent with the view that HIV spread in Myanmar might have taken place at about the same time as that in Thailand, and that multiple entries and exchanges of HIV-1 with neighboring countries are important factors contributing to the current distribution of subtypes in Myanmar.
Assuntos
HIV-1/classificação , Sequência de Aminoácidos , DNA Viral , Feminino , Heterogeneidade Genética , Infecções por HIV/epidemiologia , Soropositividade para HIV/virologia , HIV-1/genética , Humanos , Masculino , Epidemiologia Molecular , Dados de Sequência Molecular , Mianmar/epidemiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Sorotipagem , Tailândia/epidemiologiaRESUMO
To investigate the nature of recent HIV outbreaks among injecting drug users (IDUs) near the Vietnam-China border, we genetically analyzed 24 HIV-positive blood specimens from 2 northern provinces of Vietnam (Lang Son and quang Ninh) adjacent to the China border, where HIV outbreaks among IDUs were first detected in late 1996. Genetic subtyping based on gag (p17) and env (C2/V3) sequences revealed that CRF01_AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01_AE sequences among IDUs in Quang Ninh and Lang Son showed significant clustering with those found in nearby Pingxiang City of China's Guangxi Province, sharing a unique valine substitution 12 amino acids downstream of the V3 loop. This particular subtype E variant, uniquely found among IDUs in northern Vietnam and southeastern China, is designated E(v). The genetic diversity of CRF01_AE distributed in Quang Ninh (1.5 +/- 0.6%) and Pingxiang City (1.9 +/- 1.2%) was remarkably low, indicating the emerging nature of HIV spread in these areas. It is also noted that the genetic diversity of CRF01_AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01_AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01_AE had been introduced via the sexual route. The data in the present study provide additional evidence that HIV outbreaks among IDUs in northern Vietnam were caused by the recent introduction of a highly homogeneous CRF01_AE variant (E(v)) closely related to that prevailing in nearby southern China.